Useful mutations of bacterial alkaline protease

ABSTRACT

The present invention relates to mutations of a subtilisin gene which result in changes in the chemical characteristics of subtilisin enzymes. Mutations at specific nucleic acids of the subtilisin gene result in amino acid substitutions and consequently, altered enzyme function. Some of these mutant enzymes exhibit physical properties advantageous to industrial applications, particularly in the detergent industry, providing subtilisin which is more stable to oxidation, possesses greater protease activity, and exhibits improved washability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/486,846filed Jun. 7, 1995, now U.S. Pat. No. 6,506,589, which is a division ofapplication Ser. No. 07/294,241 filed Jan. 6, 1989, now abandoned andclaims priority of Danish application no. 64/88 filed Jan. 7, 1988, thecontents of which are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to mutations of the subtilisin gene whichresult in changes in the chemical characteristics of subtilisin enzyme.Mutations at specific nucleic acids of the subtilisin gene result inamino acid substitutions and consequently, altered enzyme function. Someof these mutant enzymes exhibit physical properties advantageous toindustrial applications, particularly in the detergent industry,providing subtilisin which is more stable to oxidation, possessesgreater protease activity, and exhibits improved washability.

2. Description of Related Art

Bacillus Proteases

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (See Walsh, 1979,Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco,Chapter 3). Bacteria of the Bacillus species secrete two extracellularspecies of protease, a neutral, or metalloprotease, and an alkalineprotease which is functionally a serine endopeptidase, referred to assubtilisin. Secretion of these proteases has been linked to thebacterial growth cycle, with greatest expression of protease during thestationary phase, when sporulation also occurs. Joliffe et al. (1980, J.Bacterial. 141:1199-1208) has suggested that Bacillus proteases functionin cell wall turnover.

Subtilisins

A serine protease is an enzyme which catalyses the hydrolysis of peptidebonds, in which there is an essential serine residue at the active site(White, Handler, and Smith, 1973, “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, New York, pp. 271-272).

The serine proteases have molecular weights in the 25,000 to 30,000range. They are inhibited by diisopropylfluorophosphate, but in contrastto metalloproteases, are resistant to ethylenediamine-tetra acetic acid(EDTA) (although they are stabilized at high temperatures by calciumion). They hydrolyze simple terminal esters and are similar in activityto eukaryotic chymotrypsin, also a serine protease. The alternativeterm, alkaline protease, reflects the high pH optimum of the serineproteases, from pH 9.0 to 11.0 (for review, see Priest, 1977,Bacteriological Rev. 41:711-753).

A subtilisin is a serine protease produced by Gram-positive bacteria orfungi. A wide variety of subtilisins have been identified, and the aminoacid sequences of at least eight subtilisins have been determined. Theseinclude six subtilisins from Bacillus strains, namely, subtilisin 168,subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisinamylosacchariticus, and mesentericopeptidase (Kurihara et al., 1972, J.Biol. Chem. 247:5629-5631; Stahl and Ferrari, 1984, J. Bacteriol.158:411-418; Vasantha et al., 1984, J. Bacteriol. 159:811-819, Jacobs etal., 1985, Nucl. Acids Res. 13:8913-8926; Nedkov et al., 1985, Biol.Chem. Hoppe-Seyler 366:421-430; Svendsen et al., 1986, FEBS Lett196:228-232), and two fungal subtilisins, subtilisin thermitase fromThermoactinymyces vulgaris (Meloun et al., 1985, FEBS Lett. 183:195-200)and proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol.Chem. Hoppe-Seyler 366:584-492).

Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinhave been determined which delineate the binding of substrate,transition state, products, three different protease inhibitors, anddefine the structural consequences for natural variation (Kraut, 1971,Ann. Rev. Biochem. 46:331-358). Random and site-directed mutations ofthe subtilisin gene have both arisen from knowledge of the physical andchemical properties of the enzyme and contributed information relatingto subtilisin's catalytic activity, substrate specificity, tertiarystructure, etc. (Wells et al., 1987, Proc. Natl. Acad. Sci. U.S.A.84:1219-1223; Wells et al., 1986, Phil. Trans. R. Soc. Lond. A.317:415-423; Hwang and Warshel, 1987, Biochem. 26:2669-2673; Rao et al.,1987, Nature 328:551-554).

Industrial Applications of Subtilisins

Subtilisins have found much utility in industry, particularly detergentformulations, as they are useful for removing proteinaceous stains. Tobe effective, however, these enzymes must not only possess activityunder washing conditions, but must also be compatible with otherdetergent components during storage. For example, subtilisin may be usedin combination with amylases, which are active against starches;cellulases which will digest cellulosic materials; lipases, which areactive against fats; peptidases, which are active on peptides, andureases, which are effective against urine stains. Not only must theformulation protect other enzymes from digestion by subtilisin, butsubtilisin must be stable with respect to the oxidizing power, calciumbinding properties, detergency and high pH of nonenzymatic detergentcomponents. The ability of the enzyme to remain stable in their presenceis often referred to as its washing ability or washability.

SUMMARY OF THE INVENTION

The present invention relates to mutations of the subtilisin gene, someof which result in changes in the chemical characteristics of subtilisinenzyme. Mutations are created at specific nucleic acids of thesubtilisin gene, and, in various specific embodiments, the mutantenzymes possess altered chemical properties including, but not limitedto, increased stability to oxidation, augmented proteolytic ability, andimproved washability.

The present invention also relates, but is not limited to the amino acidand DNA sequences for protease mutants derived from Bacillus lentusvariants, subtilisin 147 and subtilisin 309, as well as mutations ofthese genes and the corresponding mutant enzymes.

Site-directed mutation can efficiently produce mutant subtilisin enzymeswhich can be tailored to suit a multitude of industrial applicationsparticularly in the areas of detergent and food technology. The presentinvention relates, in part, but is not limited to, mutants of thesubtilisin 309 gene which exhibit improved stability to oxidation,augmented protease activity, and/or improved washability.

Abbreviations

A=Ala=Alanine

V=Val=Valine

L=Leu=Leucine

I=Ile=Isoleucine

P=Pro=Proline

F=Phe=Phenylalanine

W=Trp=Tryptophan

M=Met=Methionine

G=Gly=Glycine

S=Ser=Serine

T=Thr=Threonine

C=Cys=Cysteine

Y=Tyr=Tyrosine

N=Asn=Asparagine

Q=Gin=Glutamine

D=Asp=Aspartic Acid

E=Glu=Glutamic Acid

K=Lys=Lysine

R=Arg=Arginine

H=His=Histidine

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the insertion of a subset of fragments, ranging from1.5 kb to 6.5 kb in length, generated by partial digestion of Bacilluslentus strain 309 DNA with Sau 3A restriction endonuclease, into Bam HIcut plasmid pSx50. The two resulting plasmids, pSx86 and pSx88,containing the subtilisin 309 gene in opposite orientations, are alsoshown.

FIG. 2 illustrates the insertion of Bacillus lentus strain 147 DNAfragments into plasmid pSX56. Partial digestion of strain 147 DNA wasperformed using Sau 3A restriction endonuclease. Fragments ranging insize from 1.5 to 6.5 kb were then ligated into Bam HI cleaved plasmidpSX56. The product, pSX94, contains the subtilisin 147 gene.

FIG. 3 illustrates gapped duplex mutagenesis, using the method ofMorinaga et al. (1984, Biotechnology 2:636-639). It features twoplasmids, pSX93 and pSX119, both derived from puCl3. pSX93 contains anXbaI-HindIII fragment of the subtilisin 309 gene, and pSX119 containsthe remainder of the subtilisin 309 gene in an EcoRI-XbaI fragment. In(A), plasmid pSX93 is cleaved with XbaI and ClaI, and the gappedmolecules are mixed with pSX93 cut with ScaI, denatured, and allowed toreanneal so as to generate plasmids with a region of single-stranded DNAextending within the subtilisin 309 coding sequence. Ä syntheticoligonucleotide, homologous to the subtilisin 309 gene but containing amutation, is allowed to anneal to the single stranded gap, which is thenfilled in using the Klenow fragment of DNA polymerase I and T4 DNAligase. Upon replication of the plasmid, double-stranded mutants of thesubtilisin 309 gene are generated. The same procedure is performed in(B), using plasmid pSX119 and EcoRI and XbaI enzymes, to createmutations in the corresponding region of the subtilisin 309 gene.

FIG. 4 illustrates plasmid pSX92, which is a derivative of plasmidpSX62, bearing the subtilisin 309 gene. Mutated fragments (i.e.,XbaI-ClaI, XbaI-HindIII, or EcoRI-XbaI), excised from mutation plasmidpSX93 or pSX119 (see FIG. 3) using the appropriate restrictionendonucleases, were inserted into plasmid pSX92 for expression in B.subtilis strain DN 497.

FIG. 5 illustrates plasmid pSX143, which contains truncated forms ofboth subtilisin 309 and subtilisin 147 genes. In vivo recombinationbetween homologous regions of the two genes can result in activeprotease.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to mutations of the subtilisin gene, some of whichresult in changes in the chemical characteristics of subtilisin enzyme.Mutations at specific nucleic acids may be generated, and thus, forms ofsubtilisin can be designed so as to meet the needs of industrialapplication.

The invention is based, in part, upon the discovery that mutations ofspecific nucleic acids in the subtilisin gene can result in enzymes withaltered properties. In various embodiments, enzymes with improvedstability to oxidation, augmented protease activity, or improved washingability can be generated.

For purposes of clarity in description, and not by way of limitation,the invention will be described in four parts: (a) the chemicalstructure of known subtilisins and subtilisin 147 and 309; (b) methodsfor producing mutations in the subtilisin gene; (c) expression ofmutants of subtilisin and (d) screening of subtilisin mutants fordesirable chemical properties.

Chemical Structures of Known Subtilisins and Subtilisin 147 and 309

Sequence analysis of subtilisin from various sources can reveal thefunctional significance of the primary amino acid sequence, and candirect the creation of new mutants with deliberately modified functions.Comparing the amino acid sequence of different forms of subtilisin,while contrasting their physical or chemical properties, may revealspecific target regions which are likely to produce useful mutantenzymes.

The amino acid sequences of at least eight subtilisins are known. Theseinclude six subtilisins from Bacillus strains, namely, subtilisin 168,subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisinamylosacchariticus and mesentericopeptidase (Kurihara et al., 1972, J.Biol. Chem. 247:5629-5631; Stahl and Ferrari, 1984, J. Bacteriol.158:411-418; Vasantha et al., 1984, J. Bacteriol. 159:811-819; Jacobs etal., 1985, Nucl. Acids Res. 13:8913-8926; Nedkov et al., 1985, Biol.Chem. Hoppe-Seyler 366:421-430; Svendsen et al., 1986, FEBS Lett.196:228-232), and two fungal subtilisins, subtilisin thermitase fromThermoactinymyces vulgaris (Meloun et al., 1985, FEBS Lett.183:195-200), and proteinase K from Tritirichium album limber (Janny andMayer, 1985, Biol. Chem. Hoppe-Seyler 366:485-492).

In connection with this invention the amino acid and DNA sequences fortwo further serine proteases are revealed. These proteases were derivedfrom two Bacillus lentus variants, C303 and C360, which have beendeposited with NCIB and designated the accession nos. NCIB 10147 andNCIB 10309, respectively. For convenience the proteases produced bythese strains are designated subtilisin 147 and subtilisin 309,respectively, and the genes encoding these proteins are referred to asthe subtilisin 147 and 309 genes.

As used in this invention, the term “subtilisin material” refers to aproteinaceous material which contains a subtilisin as its activeingredient. As used herein, and under the definition of subtilisinmaterial, any serine protease is a subtilisin which has at least 30%,preferably 50%, and more preferably 80% amino acid sequence homologywith the sequences referenced above for subtilisin 147, subtilisin 309,subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin DY,subtilisin amylosacchariticus, mesentericopeptidase, thermitase,proteinase K and thermomycolase. These serine proteases are alsodescribed herein as “homologous serine proteases”.

Table I compares the deduced amino acid sequences of subtilisin 309,subtilisin 147, subtilisin BPN′, subtilisin Carlsberg and subtilisin 168(Spizizen, et al., 1958, Proc. Natl. Acad. Sci. U.S.A. 44:1012-1078).Table II presents the nucleic acid sequence of the subtilisin 309 gene,and Table III presents the nucleic acid sequence of the subtilisin 147gene. The sequences of subtilisin 309 or 147, or their functionalequivalents, can be used in accordance with the invention. For example,the sequences of subtilisin 309 or 147 depicted in Tables I, II or IIIcan be altered by substitutions, additions or deletions that provide forfunctionally equivalent molecules. For example, due to the degeneracy ofnucleotide coding sequences, other DNA sequences which encodesubstantially the same amino acid sequence as depicted in Table I may beused in the practice of the present invention. These include but are notlimited to nucleotide sequences comprising all or portions of thesubtilisin 309 or 147 sequences depicted in Table II or III which arealtered by the substitution of different codons that encode the same ora functionally equivalent amino acid residues within the sequence, thusproducing a silent change. For example, one or more amino acid residueswithin the sequence can be substituted by another amino acid of asimilar polarity which acts as a functional equivalent. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic and glutamic acid.

Closeness of relation can be measured by comparison of amino acidsequences. There are many methods of aligning protein sequences, but thedifferences are only manifest when the degree of relatedness is quitesmall. The methods described in Atlas of Protein Sequence and Structure,Margaret O. Dayhoff editor, Vol. 5, Supplement 2, 1976, NationalBiomedical Research Foundation, Georgetown University Medical Center,Washington, D.C., p. 3 ff., entitled SEARCH and ALIGN, definerelatedness. As is well known in the art, related proteins can differ innumber of amino acids as well as identity of each amino acid along thechain. That is, there can be deletions or insertions when two structuresare aligned for maximum identity. For example, subtilisin Carlsberg hasonly 274 amino acids while subtilisin BPN′ has 275 amino acids. Aligningthe two sequences shows that Carlsberg has no residue corresponding toAsn 56 of subtilisin BPN′. Thus the amino acid sequence of Carlsbergwould appear very different from subtilisin BPN′ unless a gap isrecorded at location 56. Therefore, one can predict with a high degreeof confidence that substituting Ser for Asn at location 218 ofsubtilisin Carlsberg will increase thermal stability provided that theresidues in Carlsberg are numbered by homology to subtilisin BPN′.

According to the invention, the sequences determined for subtilisins 309and 147 can be compared with sequences of known subtilisins (see TableI) or newly discovered subtilisins in order to deduce sites fordesirable mutations. To do this, the closeness of relation of thesubtilisins being compared must be determined.

Experiments to determine the relationship between the primary structureof subtilisin and its physical properties have revealed the significanceof the methionine-222 residue as well as the amino acids functional inthe native site, namely, aspartic acid-32, histidine-64, and serine-221.Asparagine-155 and Serine-221 are within the oxyanion binding site.Mutations at these positions are likely to diminish proteolyticactivity. According to the present invention, the amino acid sequencesof subtilisins 309 and 147 were compared with one another and with thesequences of other subtilisins (see Table II). Residues that variedbetween subtilisin 309 or 147 and other subtilisins were identified. Forexample, at residue 153, subtilisin 309 contains a serine residue,whereas subtilisin 147, subtilisin BPN′, Carlsberg and 168 contain analanine residue. Therefore, if the serine 153 residue of subtilisin 309were changed to an alanine residue, the physical properties ofsubtilisin 309 might be altered in a desired direction. Likewise,subtilisin 147 contains a serine residue at position 218, whereas theother subtilisins expressed an asparagine residue. Because subtilisin147 has improved thermal stability relative to the other subtilisins,mutating the asparagine 218 of subtilisin 309 to a serine residue mightimprove the thermal stability of subtilisin 309. As another example, itwas reasoned that, since Thr 71 is close to the active site, theintroduction of a negatively charged amino acid, such as aspartic acid,might suppress oxidative attack by electrostatic repulsion. The sitesthat are most likely to be relevant to the physical properties ofsubtilisin are those in which there is conservation of amino acidresidues between most subtilisins, for example Asp-153 and Asn-218discussed above, and also Trp-6, Arg-170, Pro-168, His-67, Met-175,Gly-219, Arg-275. By mutating the nucleic acid sequence such that anamino acid which differs from other subtilisins is substituted with anamino acid that conforms, a more stable form of subtilisin may result.

Wells et al. (1987, Proc. Natl. Acad. Sci. U.S.A. 84:1219-1223) haveused comparison of amino acid sequences and site-directed mutation toengineer subtilisin substrate specificity. The catalytic activities ofvarious subtilisins can differ markedly against selected substrates.Wells has shown that only three amino acid substitutions can cause B.amyloliquefaciens subtilisin substrate specificity to approach that ofB. licheniformis subtilisin, enzymes that differ by factors of 10-50 incatalytic efficiency in their native state. Comparison analysis betweensubtilisin 147 and 309 and other subtilisins has indicated that mutationof the following sites may alter the physical or chemical properties ofsubtilisin: 6, 9, 11-12, 19, 25, 36-38, 53-59, 67, 71, 89, 104, 111,115, 120, 121-122, 124, 128, 131, 140, 153-166, 168, 169-170, 172, 175,180, 182, 186, 187, 191, 194, 195, 199, 218, 219, 222, 226, 234-238,241, 260-262, 265, 268, or 275. Deletions occur at the following sitesin subtilisins 147 and/or 309; insertion of appropriate amino acidresidues into these sites might enhance the stability of the parentenzymes: 1, 36, 56, 159, 164-166. According to the method illustrated bythese examples, which are not limiting, a number of potential mutationsites become apparent.

TABLE I COMPARISON OF AMINO ACID SEQUENCE FOR VARIOUS PROTEASES                  10                  20                  30 a)A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V- b)*-Q-T-V-P-W-G-I-S-F-I-N-T-Q-Q-A-H-N-R-G-I-F-G-N-G-A-R-V-A-V- c)A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V- d)A-Q-T-V-P-Y-G-I-P-L-I-K-A-D-K-V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V- e)A-Q-S-V-P-Y-G-I-S-Q-I-K-A-P-A-L-H-S-Q-G-Y-T-G-S-N-V-K-V-A-V-                  40                  50                  60 a)L-D-T-G-I-*-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-Q-D- b)L-D-T-G-I-*-A-T-H-P-D-L-R-I-A-G-G-A-S-F-I-S-S-E-P-*-S-Y-H-D- c)I-D-S-G-I-D-S-S-H-P-D-L-K-V-A-G-G-A-S-M-V-P-S-E-T-N-P-F-Q-D- d)L-D-T-G-I-Q-A-S-H-P-D-L-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-T-D- e)L-D-S-G-I-D-S-S-H-P-D-L-N-V-R-G-G-A-S-F-V-A-S-E-T-N-P-Y-Q-D-                  70                  80                  90 a)G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-E-L- b)N-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-D-L- c)N-N-S-H-G-T-H-V-A-G-T-V-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-S-L- d)G-N-G-H-G-T-H-V-A-G-T-V-A-A-L-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L- e)G-S-S-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-S-P-S-A-S-L-                  100                 110                 120 a)Y-A-V-K-V-L-G-A-S-G-S-G-S-V-S-S-I-A-Q-G-L-E-W-A-G-N-N-G-M-H- b)Y-A-V-K-V-L-D-R-N-G-S-G-S-L-A-S-V-A-Q-G-I-E-W-A-I-N-N-N-M-H- c)Y-A-V-K-V-L-G-A-D-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-A-I-A-N-N-M-D- d)Y-A-V-K-V-L-N-S-S-G-S-G-T-Y-S-G-I-V-S-G-I-E-W-A-T-T-N-G-M-D- e)Y-A-V-K-V-L-D-S-T-G-S-G-Q-Y-S-W-I-I-N-G-I-E-W-A-I-S-N-N-M-D-                  130                 140                 150 a)V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V- b)I-I-N-M-S-L-G-S-T-S-G-S-S-T-L-E-L-A-V-N-R-A-N-N-A-G-I-L-L-V- c)V-I-N-M-S-L-G-G-P-S-P-S-A-A-L-K-A-A-V-D-K-A-V-A-S-G-V-V-V-V- d)V-I-N-M-S-L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A-R-G-V-V-V-V- e)V-I-N-M-S-L-G-G-P-T-G-S-A-A-L-K-T-V-V-D-K-A-V-S-S-G-I-L-V-A-                  160                 170                 180 a)A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A-N-A-M-A-V-G-A-T- b)G-A-A-G-N-T-G-R-*-Q-G-V-N-*-*-*-Y-P-A-R-Y-S-G-V-M-A-V-A-A-V- c)A-A-A-G-N-E-G-T-S-G-S-S-S-T-V-G-Y-P-G-K-Y-P-S-V-I-A-V-G-A-V- d)A-A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D-S-V-I-A-V-G-A-V- e)A-A-A-G-N-E-G-S-S-G-S-S-S-T-V-G-Y-P-A-K-Y-P-S-T-I-A-V-G-A-V-                  190                 200                 210 a)D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-G-V-N-V-Q-S-T-Y-P- b)D-Q-N-G-Q-P-P-S-F-S-T-Y-G-P-E-I-E-I-S-A-P-G-V-N-V-N-S-T-Y-T- c)D-S-S-N-Q-R-A-S-F-S-S-V-G-P-E-L-D-V-M-A-P-G-V-S-I-Q-S-T-L-P- d)D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A-P-G-A-G-V-Y-S-T-Y-P- e)N-S-S-N-Q-R-A-S-F-S-S-A-G-S-E-L-D-V-M-A-P-G-V-S-I-Q-S-T-L-P-                  220                  230                 240 a)G-S-T-Y-A-S-L-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S- b)G-N-R-Y-V-S-L-S-G-T-S-M-A-T-P-H-V-A-G-V-A-A-L-V-K-S-R-Y-P-S- c)G-N-K-Y-G-A-Y-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N- d)T-S-T-Y-A-T-L-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N- e)G-G-T-Y-G-A-Y-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-T-                  250                 260                 270 a)W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-S-T-N-L-Y-G-S-G-L-V-N-A- b)Y-T-N-N-Q-I-R-Q-R-I-N-Q-T-A-T-Y-L-G-S-P-S-L-Y-G-N-G-L-V-H-A- c)W-T-N-T-Q-V-R-S-S-L-E-N-T-T-T-K-L-G-D-S-F-Y-Y-G-K-G-L-I-N-V- d)L-S-A-S-Q-V-R-N-R-L-S-S-T-A-T-Y-L-G-S-S-F-Y-Y-G-K-G-L-I-N-V- e)W-T-N-A-Q-V-R-D-R-L-E-S-T-A-T-Y-L-G-N-S-F-Y-Y-G-K-G-L-I-N-V- a)E-A-A-T-R (SEQ ID NO: 1) b) G-R-A-T-Q (SEQ ID NO: 2) c) Q-A-A-A-Q (SEQID NO: 3) d) E-A-A-A-Q (SEQ ID NO: 4) e) Q-A-A-A-Q (SEQ ID NO: 5) a= subtilisin 309 b = subtilisin 147 c = subtilisin BPN′ d = subtilisinCarlsberg e = subtilisin 168 * = assigned deletion

Methods for Producing Mutations in Subtilisin Genes

Many methods for introducing mutations into genes are well known in theart. After a brief discussion of cloning subtilisin genes, methods forgenerating mutations in both random sites and specific sites within thesubtilisin gene will be discussed.

Cloning a Subtilisin Gene

The gene encoding subtilisin may be cloned from any Gram-positivebacteria or fungus by various methods well known in the art. First agenomic and/or cDNA library of DNA must be constructed using chromosomalDNA or messenger RNA from the organism that produces the subtilisin tobe studied. Then, if the amino acid sequence of the subtilisin is known,homologous, labelled oligonucleotide probes may be synthesized and usedto identify subtilisin-encoding clones from a genomic library ofbacterial DNA, or from a fungal cDNA library. Alternatively, a labelledoligonucleotide probe containing sequences homologous to subtilisin fromanother strain of bacteria or fungus could be used as a probe toidentify subtilisin-encoding clones, using hybridization and washingconditions of lower stringency.

Yet another method for identifying subtilisin-producing clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming protease-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for subtilisin, such as skim milk.Those bacteria containing subtilisin-bearing plasmid will producecolonies surrounded by a halo of clear agar, due to digestion of theskim milk by excreted subtilisin.

Generation of Random Mutations in the Subtilisin Gene

Once the subtilisin gene has been cloned into a suitable vector, such asa plasmid, several methods can be used to introduce random mutationsinto the gene.

One method would be to incorporate the cloned subtilisin gene, as partof a retrievable vector, into a mutator strain of Eschericia coli.

Another method would involve generating a single stranded form of thesubtilisin gene, and then annealing the fragment of DNA containing thesubtilisin gene with another DNA fragment such that a portion of thesubtilisin gene remained single stranded. This discrete, single strandedregion could then be exposed to any of a number of mutagenizing agents,including, but not limited to, sodium bisulfite, hydroxylamine, nitrousacid, formic acid, or hydralazine. A specific example of this method forgenerating random mutations is described by Shortle and Nathans (1978,Proc. Natl. Acad. Sci. U.S.A., 75:2170-2174). According to the Shortleand Nathans method, the plasmid bearing the subtilisin gene would benicked by a restriction enzyme that cleaves within the gene. This nickwould be widened into a gap using the exonuclease action of DNApolymerase I. The resulting single-stranded gap could then bemutagenized using any one of the above mentioned mutagenizing agents.

Alternatively, the subtilisin gene from a Bacillus species including thenatural promoter and other control sequences could be cloned into aplasmid vector containing replicons for both E. coli and B. subtilis, aselectable phenotypic marker and the M13 origin of replication forproduction of single-stranded plasmid DNA upon superinfection withhelper phage IR1. Single-stranded plasmid DNA containing the clonedsubtilisin gene is isolated and annealed with a DNA fragment containingvector sequences but not the coding region of subtilisin, resulting in agapped duplex molecule. Mutations are introduced into the subtilisingene either with sodium bisulfite, nitrous acid or formic acid or byreplication in a mutator strain of E. coli as described above. Sincesodium bisulfite reacts exclusively with cytosine in a single-strandedDNA, the mutations created with this mutagen are restricted only to thecoding regions. Reaction time and bisulfite concentration are varied indifferent experiments such that from one to five mutations are createdper subtilisin gene on average. Incubation of 10 micrograms of gappedduplex DNA in 4 M Na-bisulfite, pH. 6.0, for 9 minutes at 37° C. in areaction volume of 400 microliters, deaminates about 1% of cytosines inthe single-stranded region. The coding region of mature subtilisincontains about 200 cytosines, depending on the DNA strand.Advantageously, the reaction time is varied from about 4 minutes (toproduce a mutation frequency of about one in 200) to about 20 minutes(about 5 in 200).

After mutagenesis the gapped molecules are treated in vitro with DNApolymerase I (Klenow fragment) to make fully double-stranded moleculesand to fix the mutations. Competent E. coli are then transformed withthe mutagenized DNA to produce an amplified library of mutantsubtilisins. Amplified mutant libraries can also be made by growing theplasmid DNA in a Mut D strain of E. coli which increases the range ofmutations due to its error prone DNA polymerase.

The mutagens nitrous acid and formic acid may also be used to producemutant libraries. Because these chemicals are not as specific forsingle-stranded DNA as sodium bisulfite, the mutagenesis reactions areperformed according to the following procedure. The coding portion ofthe subtilisin gene is cloned in M13 phage by standard methods andsingle stranded phage DNA prepared. The single-stranded DNA is thenreacted with 1 M nitrous acid pH 4.3 for 15-60 minutes at 23° C. or 2.4M formic acid for 1-5 minutes at 23° C. These ranges of reaction timesproduce a mutation frequency of from 1 in 1000 to 5 in 1000. Aftermutagenesis, a universal primer is annealed to the M13 DNA and duplexDNA is synthesized using the mutagenized single stranded DNA as atemplate so that the coding portion of the subtilisin gene becomes fullydouble-stranded. At this point the coding region can be cut out of theM13 vector with restriction enzymes and ligated into an unmutagenizedexpression vector so that mutations occur only in the restrictionfragment (Myers et al., 1985, Science 229:242-257).

By yet another method, mutations can be generated by allowing twodissimilar forms of subtilisin to undergo recombination in vivo.According to this method, homologous regions within the two genes leadto a cross-over of corresponding regions resulting in the exchange ofgenetic information. The generation of hybrid amylase moleculesaccording to this technique is fully described in U.S. application Ser.No. 67,992, filed on Jun. 29, 1987, which is fully incorporated hereinby reference. An example of a plasmid which can generate hybrid forms ofsubtilisin is depicted in FIG. 5. Both the subtilisin 309 and 147 genes,incorporated into plasmid pSX143, are truncated, and therefore cannotthemselves lead to subtilisin expression. However, if recombinationoccurs between the two genes so as to correct the defect produced bytruncation, i.e., the N terminal region of the subtilisin 309 genebecomes linked to the C terminal region of the subtilisin 147 gene, thenactive, mutant subtilisin can be produced. If pSX143 is incorporatedinto a protease-negative strain of bacteria, and then bacteria thatdevelop a protease positive phenotype are selected, then variousmutants, subtilisin 309/147 chimeras, can be identified.

Generation of Site Directed Mutations in the Subtilisin Gene

Once the subtilisin gene has been cloned, and desirable sites formutation identified, these mutations can be introduced using syntheticoligo nucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites; mutant nucleotides are insertedduring oligonucleotide synthesis. In a preferred method, a singlestranded gap of DNA, bridging the subtilisin gene, is created in avector bearing the subtilisin gene. Then the synthetic nucleotide,bearing the desired mutation, is annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in by DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described in Morinaga etal. (1984, Biotechnology 2:636-639). According to Morinaga et al., afragment within the gene is removed using restriction endonuclease. Thevector/gene, now containing a gap, is then denatured and hybridized tovector/gene which, instead of containing a gap, has been cleaved withanother restriction endonuclease at a site outside the area involved inthe gap. A single-stranded region of the gene is then available forhybridization with mutated oligonucleotides, the remaining gap is filledin by the Klenow fragment of DNA polymerase I, the insertions areligated with T4 DNA ligase, and, after one cycle of replication, adouble-stranded plasmid bearing the desired mutation is produced. TheMorinaga method obviates the additional manipulation of construction newrestriction sites, and therefore facilitates the generation of mutationsat multiple sites. U.S. Pat. No. 4,760,025, by Estelle et al., issuedJul. 26, 1988, is able to introduce oligonucleotides bearing multiplemutations by performing minor alterations of the cassette, however, aneven greater variety of mutations can be introduced at any one time bythe Morinaga method, because a multitude of oligonucleotides, of variouslengths, can be introduced.

Expression of Subtilisin Mutants

According to the invention, a mutated subtilisin gene produced bymethods described above, or any alternative methods known in the art,can be expressed, in enzyme form, using an expression vector. Anexpression vector generally falls under the definition of a cloningvector, since an expression vector usually includes the components of atypical cloning vector, namely, an element that permits autonomousreplication of the vector in a microorganism independent of the genomeof the microorganism, and one or more phenotypic markers for selectionpurposes. An expression vector includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene. To permit the secretion ofthe expressed protein, nucleotides encoding a “signal sequence” may beinserted prior to the coding sequence of the gene. For expression underthe direction of control sequences, a target gene to be treatedaccording to the invention is operably linked to the control sequencesin the proper reading frame. Promoter sequences that can be incorporatedinto plasmid vectors, and which can support the transcription of themutant subtilisin gene, include but are not limited to the prokaryoticbeta-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad.Sci. U.S.A. 75:3727-3731) and the tac promoter (DeBoer, et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Further references can also befound in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242:74-94.

According to one embodiment B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term “control sequences” as defined above is intended to include asignal sequence, when it is present.

Screening of Mutant Subtilisins

For screening mutants, transformed B. subtilis can be cultivated in thepresence of a filter material (such as nitrocellulose) to which thesecreted expression product (e.g., enzyme) binds. In order to screen foran expression product having a desired characteristic, filter boundexpression product is subjected to conditions which distinguishexpression product of interest from wild-type expression product. Forexample, the filter-bound expression product can be subjected toconditions which would inactivate a wild-type product. Preserved enzymeactivity following adverse treatment suggests that the mutation confersenhanced stability on the enzyme, and is therefore a useful mutation.

In one embodiment of the invention, screening for stable variants isaccomplished using a protease deficient B. subtilis strain transformedwith the variant plasmid and plated out as follows: a nitrocellulosefilter is placed on a nutrient base in a petri dish, and a celluloseacetate filter is placed on top of the nitrocellulose. Colonies aregrown on the cellulose acetate, and protease from individual colonies issecreted through the cellulose acetate onto the nitrocellulose filterwhere it is stably bound. Protease from hundreds of colonies is bound toa single filter allowing subsequent screening of thousands of differentvariants by processing multiple filters.

To identify colonies producing subtilisin of enhanced thermal stability,the filters can be incubated in buffer solutions at temperatures whichwould inactivate substantially all wild-type activity. Variants ofenhanced stability or activity retain activity after this step. Thesuitably treated filter then is soaked in a solution containingTosyl-L-Arg methyl ester (TAME), Benzoly-Arg-ethyl-ester (BAEE),Acetyl-Tyr-ethyl-ester (ATEE) (Sigma) or similar compounds. BecauseTAME, BAEE, and ATEE are substrates for the proteases they are cleavedin zones on the filter containing variant subtilisins which remainactive after treatment. As cleavage occurs, protons are released in thereaction and cause phenol red to change in color from red to yellow inareas retaining protease activity.

This procedure can be used to screen for different classes of variantswith only slight modifications. For example, the filters could betreated at high temperature, at high pH, with denaturants, oxidizingagents, or under other conditions which normally inactivate an enzymesuch as a protease to find resistant variants. Variants with alteredsubstrate specificity could be screened by replacing TAME, BAEE, or ATEEwith other substrates which are normally not cleaved by wild-typesubtilisin.

Once a variant of enhanced stability is identified by screening, thecolony from which the variant is derived is isolated and the alteredsubtilisin is purified. Experiments can be performed on the purifiedenzyme to determine conditions of stability towards oxidation, thermalinactivation, denaturation temperature, kinetic parameters as well asother physical measurements. The altered gene can also be sequenced todetermine the amino acid changes responsible for the enhanced stability.Using this procedure, variants with increased washing abilities havebeen isolated.

EXAMPLES Site-specific Mutation of the Subtilisin Gene Generates MutantsWith Useful Chemical Characteristics Materials and Methods

Bacterial Strains

B. subtilis 309 and 147 are variants of Bacillus lentus, deposited withthe NCIB and accorded the accession numbers NCIB 10147 and NCIB 10309,and described in U.S. Pat. No. 3,723,250, issued Mar. 27, 1973, andfully incorporated herein by reference herein. B. subtilis DN 497 isdescribed in U.S. application Ser. No. 039,298 filed Apr. 17, 1987,which is also fully incorporated herein by reference, and is an aro⁺transformant of RUB 200 with chromosomal DNA from SL 438, a sporulationand protease deficient strain obtained from Dr. Kim Hardy of Biogen. E.coli MC 1000 r⁻m⁺ (Casa-daban, M. J. and Cohen, S. N. (1980), J. Mol.Biol. 138:179-207, was made r⁻m⁺ by conventional methods and is alsodescribed in U.S. application Ser. No. 039,298, supra.

Plasmids

pSX50 (described in U.S. application Ser. No. 039,298, supra) is aderivative of plasmid pDN 1050, comprising the promoter-operator P₁O₁,the B. pumilus xyn B gene and the B. subtilis xyl R gene.

pSX65 (described in U.S. application Ser. No. 039,298, supra) is aderivative of plasmid pDN 1050, comprising the promoter-operator P₂O₂,the B. pumilus xyn B gene, and the B. subtilis xyl R gene.

pSX93, shown in FIG. 3A, is puCl3 (Vieira and Messing, 1982, Gene19:259-268) comprising a 0.7 kb XbaI-Hind III fragment of the subtilisin309 gene including the terminator inserted in a polylinker sequence.

pSX119 is pUC13 harboring an EcoRI-XbaI fragment of the subtilisin 309gene inserted into the polylinker.

pSX62 (described in U.S. application Ser. No. 039,298, supra) is aderivative of pSX52 (ibid), which comprises a fusion gene between thecalf prochymosin gene and the B. pumilus xyn B gene inserted into pSX50(supra). pSX62 was generated by inserting the E. coli rrn B terminatorinto pSX52 behind the prochymosin gene.

pSX92 was produced by cloning the subtilisin 309 gene into plasmid pSX62(supra) cut at ClaI and HindIII and filled prior to the insertion of thefragments DraI-NheI and NheI-HindIII from the cloned subtilisin 309gene.

Purification of Subtilisins

The procedure relates to a typical purification of a 10 liter scalefermentation of subtilisin 147, subtilisin 309 or mutants thereof.

Approximately 8 liters of fermentation broth were centrifuged at 5000rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to adsorption on aBacitracin affinity column at pH 7. The protease was eluted from theBacitracin column using 25% 2-propanol and 1 M sodium chloride in abuffer solution with 0.01 M dimethylglutaric acid, 0.1 M boric acid and0.002 M calcium chloride adjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 M dimethylglutaricacid, 0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column werecombined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium chlorideadjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof subtilisin 147).

In a final purification step protease containing fractions from the CMSepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81P membrane (from the DanishSugar Factories Inc.).

Subtillisin 309 and mutants

Met 222 to Ala

Gly 195 to Glu

Asn 218 to Ser

Arg 170 to Tyr

Gly 195 to Glu, Arg 170 to Tyr

Gly 195 to Glu, Met 222 to Ala

were purified by this procedure.

Oligonucledotide Synthesis

All mismatch primers were synthesized on an Applied Biosystems 380 A DNAsynthesizer and purified by polyacrylamide gel electrophoresis (PAGE).

Determination of Oxidation Stability

The purified enzyme is diluted to an enzyme content of approximately 0.1mg/ml in 0.01 M dimethylglutaric acid pH 7 and in the same buffer with0.01 M peracetic acid (pH 7).

Both sets of dilutions were heated to 50° C. for 20 minutes. Proteolyticactivity was measured in the dilutions before and after the heattreatment.

Assay for Proteolytic Activity OPA-Casein Method

Proteolytic activity was determined using casein as the substrate. OneCasein Protease Unit (CPU) is defined as the amount of enzyme liberating1 millimole of primary amino groups (determined by comparison with aserine standard) per minute under standard conditions, i.e., incubationfor 30 minutes at 25° C. and pH 9.5.

A 2% (w/v) solution of casein (Hammarstein, supplied by Merck A. G.,West Germany) was prepared with the Universal Buffer described byBritton and Robinson (Journ. Chem. Soc. 1931, p. 1451), adjusted to pH9.5.

Two ml of substrate solution was preincubated in a water bath for 10minutes at 25° C. One ml of enzyme solution containing about 0.2-0.3CPU/ml of Britton-Robinson buffer (pH 9.5), was added. After 30 minutesof incubation at 25° C. the reaction was terminated by the addition of astopping agent (5 ml of a solution containing trichloroacetic acid (17.9g), sodium acetate (29.9 g), and acetic acid (19.8 g), filled up to 500ml with deionized water). A blank was prepared in the same manner as thetest solution, except that the stopping agent was added prior to theenzyme solution.

The reaction mixtures were kept for 20 minutes in the water bath,whereupon they were filtered through Whatman® 42 paper filters.

Primary amino groups were determined by their color development witho-phthaldialdehyde (OPA).

Disodium tetraborate decahydrate (7.62 g) and sodium dodecylsulfate (2.0g) was dissolved in 150 ml of water. OPA (160 mg) dissolved in 4 ml ofmethanol was then added together with 400 microliters ofbeta-mercaptoethanol, whereafter the solution was made up to 200 ml withwater.

To the OPA reagent (3 ml) was added 40 microliters of theabove-mentioned filtrates with mixing. The optical density (OD) at 340nm was measured after about 5 minutes.

The OPA test was also performed with a serine standard containing 10 mgof serine in 100 ml of Britton-Robinson buffer (pH 9.5). The buffer wasused as a blank.

The protease activity was calculated from the optical densitymeasurements by means of the following formula:

CPU/g of enzyme solution=[OD _(t) −OD _(b))×C _(Ser) ×Q]/[(OD _(Ser) −OD_(B))×MW _(Ser) ×t _(i) ]CPU/g of enzyme preparation=CPU/ml: b

wherein OD_(t), OD_(b), OD_(Ser) and OD_(B) are the optical density ofthe test solution, blank, serine standard, and buffer, respectively,C_(Ser) is the concentration of serine in mg/ml in the standard,MW_(Ser) is the molecular weight of serine, Q is the dilution factor (inthis instance equal to 8) for the enzyme solution, and t_(i) is theincubation time in minutes.

In the following Table V, results from the above assay are shownrelative to the parent enzyme.

Assay for Washability

Test cloths (7 cm×7 cm, approximately 1 g) were produced by passingdesized cotton (100% cotton, DS 71) Cloth through the vessel in a MathisWashing and Drying Unit type TH (Werner Mathis A G, Zurich, Switzerland)containing spinach juice (produced from fresh spinach) and then throughthe pressure roll of the machine in order to remove excess spinachjuice.

Finally the cloth was dried in a strong air stream at room temperature,stored at room temperature for 3 weeks, and subsequently kept at −18° C.prior to use.

The tests were performed in a Terg-O-tometer test washing machine(described in Jay C. Harris “Detergency Evaluation and Testing”,Interscience Publishers Ltd., 1954, p.60-61) isothermally for 10 minutesat 100 rpm. As detergent the following standard powder detergent wasused:

Nansa S 80 0.40 g/l AE, Berol 0 65 0.15 g/l Soap 0.15 g/l STPP 1.75 g/lSodium silicate 0.40 g/l CMC 0.05 g/l EDTA 0.01 g/l Na₂SO₄ 2.10 g/lPerborate 1.00 g/l TAED 0.10 g/l

TAED=N,N,N′,N″-tetraacetyl-ethylene diamine; pH was adjusted with 4 NNAOH to 9.5. The water used was ca. 9° GH (German Hardness).

Tests were performed at enzyme concentrations of: 0, 0.05 CPU/1, and 0.1CPU/1, and two independent sets of tests were performed for each of themutants.

Eight cloths were used for each testing using one beaker (800 ml) ofdetergent. Of the cloths, four were clean and four were stained withspinach juice. Subsequent to the washing the cloths were flushed inrunning water for 25 minutes in a bucket.

The cloths were then air dried overnight (protected against day light)and the remission, R, determined on an E1REPHO 2000 spectrophotometerfrom Datacolor S.A., Dietkikon, Switzerland at 460 nm.

As a measure of the washing ability differential remission, Delta R, wasused, Delta R being equal to the remission after wash with enzyme addedminus the remission after wash with no enzyme added.

Assay for Thermostability

The same procedure as above for washability was used for estimating thethermostability of the mutants produced, by performing the test attemperatures of 40° C. and 60° C., respectively.

Results

Cloning of the Subtilisin 309 and 147 Genes

Chromosomal DNA from the “309” strain was isolated by treating a cellsuspension with Lysozyme for 30 minutes at 37° C., and then with SDS for5 minutes at 60° C. Subsequently, the suspension was extracted withphenolchloroform (50:50), precipitated with ethanol, and the precipitateredissolved in TE. This solution was treated with RNase for 1 hour at37° C.

Approximately 30 micrograms of the chromosomal DNA was partiallydigested with restriction enzyme Sau 3A (New England Biolabs) andfragments from about 1.5 kb to about 6.5 kb were isolated on DEAEcellulose paper from a 1% agarose gel (the subtilisin gene in otherspecies is approximately 1.2 kb in length).

As outlined in FIG. 1 the fragments were annealed and ligated to BamHIcut plasmid pSX50 (described in U.S. patent application Ser. No. 039,298filed Apr. 17, 1987, is which is hereby included for reference). Theplasmids were then transformed into competent B. subtilis DN 497.

The cells were then spread on LB agar plates with 10 mM phosphate pH 7,6 micrograms/ml chloramphenicol, and 0.2% xylose to induce thexyn-promoter in the plasmid. The plates also contained 1% skim milk sothe protease producing transformants could be detected by the clear halowhere the skim milk had been degraded.

Protease expressing clones were produced at a frequency of 10⁻⁴. Twoclones were found that harbored plasmids carrying the gene forsubtilisin 309, pSX86 and pSX88. The gene was then sequenced using themethod of Maxam and Gilbert. The deduced nucleotide sequence ofsubtilisin 309 is presented in Table II.

TABLE II THE SUBTILISIN 309 GENE Signal ATGAAGAAACCG TTGGGGAAAATTGTCGCAAGCACC GCACTACTCATT TCTGTTGCTTTT 1                     PROAGTTCATCGATC GCATCGGCTGCT GAAGAAGCAAAA GAAAAATATTTA ATTGGCTTTAAT                       82 GAGCAGGAAGCT GTCAGTGAGTTT GTAGAACAAGTAGAGGCAAATGAC GAGGTCGCCATT CTCTCTGAGGAA GAGGAAGTCGAA ATTGAATTGCTTCATGAATTTGAA ACGATTCCTGTT TTATCCGTTGAG TTAAGCCCAGAA GATGTGGACGCGCTTGAACTCGAT CCAGCGATTTCT                                    MatureTATATTGAAGAG GATGCAGAAGTA ACGACAATGGCG CAATCAGTGCCA TGGGGAATTAGC                                   334 CGTGTGCAAGCC CCAGCTGCCCATAACCGTGGATTG ACAGGTTCTGGT GTAAAAGTTGCT GTCCTCGATACA GGTATTTCCACTCATCCAGACTTA AATATTCGTGGT GGCGCTAGCTTT GTACCAGGGGAA CCATCCACTCAAGATGGGAATGGG CATGGCACGCAT GTGGCCGGGACG ATTGCTGCTTTA AACAATTCGATTGGCGTTCTTGGC GTAGCGCCGAGC GCGGAACTATAC GCTGTTAAAGTA TTAGGGGCGAGCGGTTCAGGTTCG GTCAGCTCGATT GCCCAAGGATTG GAATGGGCAGGG AACAATGGCATGCACGTTGCTAAT TTGAGTTTAGGA AGCCCTTCGCCA                                          XbaI AGTGCCACACTT GAGCAAGCTGTTAATAGCGCGACT TCTAGAGGCGTT CTTGTTGTAGCG GCATCTGGGAAT TCAGGTGCAGGCTCAATCAGCTAT CCGGCCCGTTAT GCGAACGCAATG GCAGTCGGAGCT ACTGACCAAAACAACAACCGCGCC AGCTTTTCACAG TATGGCGCAGGG CTTGACATTGTC GCACCAGGTGTAAACGTGCAGAGC ACATACCCAGGT TCAACGTATGCC                  ClaIAGCTTAAACGGT ACATCGATGGCT ACTCCTCATGTT GCAGGTGCAGCA GCCCTTGTTAAACAAAAGAACCCA TCTTGGTCCAAT GTACAAATCCGC AATCATCTAAAG AATACGGCAACGAGCTTAGGAAGC ACGAACTTGTAT GGAAGCGGACTT GTCAATGCAGAA GCGGCAACACGC StopTAA (SEQ ID NO: 6) 1141

The same procedure as above was used for the cloning of the subtilisin147 gene except that the DNA fragments were ligated into the plasmidpSXS6 (also described in U.S. application Ser. No. 039,298 supra), whichas indicated in FIG. 2 instead of the xyn promoter harbors the xylpromoter. One clone was found harboring a plasmid, pSX94, carrying thegene for subtilisin 147. The sequence for this gene is shown in TableIII below.

TABLE III THE SUBTILISIN 147 GENE Signal ATGAGACAAAGT CTAAAAGTTATGGTTTTGTCAACA GTGGCATTGCTT TTCATGGCAAAC 1            Pro CCAGCAGCAGCAGGCGGGGAGAAA AAGGAATATTTG ATTGTCGTCGAA CCTGAAGAAGTT              73TCTGCTCAGAGT GTCGAAGAAAGT TATGATGTGGAC GTCATCCATGAA TTTGAAGAGATTCCAGTCATTCAT GCAGAACTAACT AAAAAAGAATTG AAAAAATTAAAG AAAGATCCGAAC                                          Mature GTAAAAGCCATCGAAGAGAATGCA GAAGTAACCATC AGTCAAACGGTT CCTTGGGGAATT                                          280 TCATTCATTAAT ACGCAGCAAGCGCACAACCGCGGT ATTTTTGGTAAC GGTGCTCGAGTC GCTGTCCTTGAT ACAGGAATTGCTTCACACCCAGAC TTACGAATTGCA GGGGGAGCGAGC TTTATTTCAAGC GAGCCTTCCTATCATGACAATAAC GGACACGGAACT CACGTGGCTGGT ACAATCGCTGCG TTAAACAATTCAATCGGTGTGCTT GGTGTACGACCA TCGGCTGACTTG TACGCTCTCAAA GTTCTTGATCGGAATGGAAGTGGT TCGCTTGCTTCT GTAGCTCAAGGA ATCGAATGGGCA ATTAACAACAACATGCACATTATT AATATGAGCCTT GGAAGCACGAGT GGTTCTAGCACG TTAGAGTTAGCTGTCAACCGAGCA AACAATGCTGGT ATTCTCTTAGTA GGGGCAGCAGGT AATACGGGTAGACAAGGAGTTAAC TATCCTGCTAGA TACTCTGGTGTT ATGGCGGTTGCA GCAGTTGATCAAAATGGTCAACGC GCAAGCTTCTCT ACGTATGGCCCA GAAATTGAAATT TCTGCACCTGGTGTCAACGTAAAC AGCACGTACACA GGCAATCGTTAC GTATCGCTTTCT GGAACATCTATGGCAACACCACAC GTTGCTGGAGTT GCTGCACTTGTG AAGAGCAGATAT CCTAGCTATACGAACAACCAAATT CGCCAGCGTATT AATCAAACAGCA ACGTATCTAGGT TCTCCTAGCCTTTATGGCAATGGA TTAGTACATGCT GGACGTGCAACA   Stop CAATAA (SEQ ID NO: 7)  1084

Generation of Site-Specific Mutations of the Subtilisin 309 Gene

Site specific mutations were performed by the method of Morinaga et al.(Biotechnology, supra). The following oligonucleotides were used forintroducing the mutations:

a) Gly-195-Glu: A 27-mer mismatch primer, Nor-237, which also generatesa novel SacI restriction site (SEQ ID NO: 8) 5′CACAGTATGGGCGCAGGGCTTGACATTGTCGCACCAGG 3′ NOR-237 (SEQ ID NO: 9) 5′GTATGGCGCAGAGCTCGACATTTGTCGC 3′        SacI b) Gly-195-Asp: A 23-mermismatch primer, NOR-323, which also ge- nerates a novel BglII site (SEQID NO: 10)        AT 5′ CACAGTATGGGCGCAGGGCTTGACATTGTC 3′ (SEQ ID NO:11)    3′ CATACCGCGTCTAGAACTGTAAC 5′          BglII c) Met-222-Cys: A24-mer mismatch primer, NOR-236 (SEQ ID NO: 12)       ClaI  5′AGCTTAAACGGTACATCGATGGCTACTCCTCATGTT 3′ NOR-236 (SEQ ID NO: 13) 5′ACGGTACATCGTGCGCTACTCCTC 3′ d) Met-222-Ala: 22-mer mismatch primer,NOR-235 (SEQ ID NO: 14)        ClaI 5′AGCTTAAACGGTACATCGATGGCTACTCCTCATGTT 3′ NOR-235 (SEQ ID NO: 15) 5′CGGTACATCGGCGGCTACTCCT 3′ Both of these primers destroy the unique dialsite. e) Ser-153-Ala: An 18-mer mismatch primer, NOR-324, which also ge-nerates a novel PvuII site (SEQ ID NO: 16)        G 5′CTTGTAGCGGCATCTGGGAATTCAGGT 3′ NOR-324 (SEQ ID NO: 17) 3′CATCGCCGTCGACCCTTA 5′      PvuII f) Asn-218-Ser: A 23-mer mismatchprimer, NOR-325, which also ge- nerates a novel MspI site (SEQ ID NO:18)         TC 5′ TATGCCAGCTTAAACGGTACATCGATG 3′ NOR-324 (SEQ ID NO: 19)3′ TACGGTCGAATAGGCCATGTAGC 5′        MspI g) Thr-71-Asp: A 23-mermismatch primer, HOR-483, (SEQ ID NO: 20)        GAC 5′TGTGGCCCGGGACGATTGCTGCTT 3′ NOR-483 (SEQ ID NO: 21) 3′ACACCGGCCCCCTGTAACGACGAA 5′ h) Met-222-Cys and Gly-219-Cys: A 32-mermismatch, NOR-484, (SEQ ID NO: 22)       T    TGT 5′CAGCTTAAACGGTACATCGATGGCTACTCCTC 3′ NOR-484 (SEQ ID NO: 23)      219   222 3′ GTCGAATTTGACATGTAGCACACGATGAGGAG 5′ i+j) Gly-195-Gluand Met-222-Ala or Met-222- Cys: For these double mutants combinationsof NOR-237 and NOR-235 or NOR-236 were performed by joining the singlemutant DNA-fragments. k) Ser-153-Ala and Asn-218-Ser: A combination ofNOR-324 and NOR-325 was performed in analogy with the above.

Gapped duplex mutagenesis was performed using the plasmid pSX93 astemplate. pSX93 is shown in FIGS. 3A and 3B, and is pUC13 (Vieira, J.and Messing, J., 1982, Gene 19: 259-268) harboring an 0.7 kbXbaI-HindIII fragment of the subtilisin 309 gene including theterminator inserted in the polylinker. The terminator and the HindIIIsite are not shown in Table II.

For the introduction of mutations in the N-terminal part of the enzymethe plasmid pSX119 was used. pSX119 is pUC13 harboring an EcoRI-XbaIfragment of the subtilisin 309 gene inserted into the polylinker. Thetemplates pSX93 and pSX119 thus cover the whole of the subtilisin 309gene.

The mutations a), b), and e) were performed by cutting pSX93 with XbaIand ClaI as indicated in FIG. 3A; c), d), f), and h) were performed bycutting pSX93 with XbaI and HindIII as indicated in FIG. 3B.

Mutation g) was performed correspondingly in pSX119 by cutting withEcoRI and XbaI.

The double mutants i) and j) were produced by cutting the 0.7 kbXba-HindIII fragment from a) partially with HgiAI (HgiAI also cuts inSacI, which was introduced by the mutation). This 180 bp XbaI-HgiAIfragment and the 0.5 kb HgiAI fragment from the c) and d) mutants,respectively, were ligated to the large HindIII-XbaI fragment frompSX93.

The double mutant k) was produced as above by combining mutants e) andf).

Subsequent to annealing, filling and ligation the mixture was used totransform E. coli MC 1000 r⁻m⁺. Mutants among the transformants werescreened for by colony hybridization as described in Vlasuk et al.,1983, J. Biol. Chem., 258:7141-7148 and in Vlasuk, G. P. and Inouye, S.,p. 292-303 in ‘Experimental Manipulation of Gene Expression’ Inouye, M.(ed.) Academic Press, New York. The mutations were confirmed by DNAsequencing.

Expression of Mutant Subtilisins

Subsequent to a sequence confirmation of the correct mutation themutated DNA fragments were inserted into plasmid pSX92, which was usedfor producing the mutants.

Plasmid pSX92 is shown in FIG. 4 and was produced by cloning thesubtilisin 309 gene into plasmid pSX62 cut at ClaI, filled in with theKlenow fragment of DNA polymerase I, and cut with HindIII prior to theinsertion of the fragments DraI-NheI and NheI-HindIII from the clonedsubtilisin 309 gene.

To express the mutants the mutated fragments (XbaI-ClaI, XbaI-HindIII,or EcoRI-XbaI) were excised from the appropriate mutation plasmid pSX93or pSX119, respectively, and inserted into pSX92.

The mutated pSX92 was then used to transform B. subtilis strain DN497,which was then grown in the same medium and under the same conditions asused for the cloning of the parent gene.

After appropriate growth the mutated enzymes were recovered andpurified.

Oxidation Stability of Mutant Subtilisins

The mutants a) and d) were tested for their oxidation stability in 0.01M peracetic acid after 20 minutes at 50° C. and pH 7. The parent strainNCIB 10309 protease was used as reference.

The results are indicated in Table IV below, which presents the residualproteolytic activity in the heat treated samples relative to samplesuntreated by oxidant or heat.

TABLE IV Oxidation Stability Towards Peracetic Acid Residual Activityafter 20 min. at 50° C. Enzyme without oxidant with oxidant Subtilisin309 89% 48% mutant a 83% 45% mutant d 92% 93%

It is concluded that mutant d (Met 222 to Ala) exhibits superioroxidation stability relative to the parent enzyme and mutant a.

All the mutants except g) and h) have also been tested qualitatively in100-500 ppm hypochlorite at room temperature and 35° C., pH 6.5 and 9.0,for from 15 minutes to 2 hours.

These tests showed that mutants c), d), i), and j) (all Met-222) couldresist 3-5 times more hypochlorite than the other mutants.

When tested in a liquid detergent of the usual built type it was foundthat mutant f) exhibited superior stability compared to both the othermutants and the “parent” enzyme.

Proteolytic Activity of Mutant Subtilisins

The proteolytic activity of various mutants was tested against casein asprotein substrate, according to methods detailed supra. The results arepresented in Table V.

From the table it is seen that mutant a) exhibits enhanced activitycompared to the parent. It is also seen that the Met-222 mutants havelower activity than the parent, but due to their improved oxidationstability their application in detergent compositions containingoxidants is not precluded.

TABLE V Proteolytic Activity of Mutant Subtilisins Mutant RelativeActivity None 100 a) 120 b) 100 c) 30 d) 20 e) 100 f) 100 i) 20 j) 30

Washability of Mutant Subtilisins

The washability of various mutants was tested against spinach juiceaccording to methods detailed supra. The results are presented in TableVI.

From the table it is seen that all of the tested mutants exhibited animproved washing ability compared to the parent enzyme, and that mutantsc), d), i), and j) are markedly superior.

TABLE VI Washability of Mutant Delta R Mutant Concentration (CPU/l) 0.050.1 none 14.40 20.4 a) 18.80 21.5 b) 16.90 19.7 c) 21.80 23.8 d) 22.2023.4 e) 15.40 21.8 f) 16.60 19.3 i) 21.60 22.1 j) 20.60 22.6 95%confidence interval: +/− 0.9

Thermostability of Mutant Subtilisins

The thermostability of mutant f) was tested against the wild type enzymeby using the washability test at 40° C. and 60° C., respectively. Theresults are shown in Table VII.

From the table it is seen that mutant f) at 60° C. shows a much improvedwashability compared to the wild type enzyme, whereas at 40° C. thewashability of mutant f) is only slightly better than the wild typeenzyme.

TABLE VII Washability at Different Temperatures Delta R MutantConcentration (CPU/l) 0.05 0.1 none (40° C.) 14.40 20.4 f) (40° C.)16.60 19.3 none (60° C.) 15.10 24.9 f) (60° C.) 30.40 31.3 95%confidence interval +/− 0.9 (40° C.) and +/− 0.7 (60° C.)

Discussion

Subtilisin genes were cloned from the 147 and 309 variants of thebacterium Bacillus lentus, and the cloned genes were sequenced. Bycomparing the deduced amino acid sequences of subtilisins 147 and 309one with the other and with sequences of other subtilisins, sites which,upon mutation, might alter the physical properties of the parent enzymewere identified. Site-directed mutagenesis was used to generatemutations at several of these sites in the subtilisin 309 gene. Theresulting mutant enzymes were then expressed in a Bacillus strain, andtested against various physical and chemical parameters. Several of themutants were shown to have improved stability to oxidation, increasedproteolytic ability, or improved washability when compared withsubtilisin 309. These mutants exhibit properties desirable in enzymescomprised in detergent compositions.

23 1 269 PRT Bacillus 1 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val GlnAla Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val LysVal Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn IleArg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp GlyAsn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr AlaVal Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile AlaGln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala AsnLeu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln AlaVal Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala SerGly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg TyrAla Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn ArgAla Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala ProGly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala SerLeu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 AlaAla Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 2268 PRT Bacillus 2 Gln Thr Val Pro Trp Gly Ile Ser Phe Ile Asn Thr GlnGln Ala His 1 5 10 15 Asn Arg Gly Ile Phe Gly Asn Gly Ala Arg Val AlaVal Leu Asp Thr 20 25 30 Gly Ile Ala Thr His Pro Asp Leu Arg Ile Ala GlyGly Ala Ser Phe 35 40 45 Ile Ser Ser Glu Pro Ser Tyr His Asp Asn Asn GlyHis Gly Thr His 50 55 60 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser IleGly Val Leu Gly 65 70 75 80 Val Ala Pro Ser Ala Asp Leu Tyr Ala Val LysVal Leu Asp Arg Asn 85 90 95 Gly Ser Gly Ser Leu Ala Ser Val Ala Gln GlyIle Glu Trp Ala Ile 100 105 110 Asn Asn Asn Met His Ile Ile Asn Met SerLeu Gly Ser Thr Ser Gly 115 120 125 Ser Ser Thr Leu Glu Leu Ala Val AsnArg Ala Asn Asn Ala Gly Ile 130 135 140 Leu Leu Val Gly Ala Ala Gly AsnThr Gly Arg Gln Gly Val Asn Tyr 145 150 155 160 Pro Ala Arg Tyr Ser GlyVal Met Ala Val Ala Ala Val Asp Gln Asn 165 170 175 Gly Gln Pro Pro SerPhe Ser Thr Tyr Gly Pro Glu Ile Glu Ile Ser 180 185 190 Ala Pro Gly ValAsn Val Asn Ser Thr Tyr Thr Gly Asn Arg Tyr Val 195 200 205 Ser Leu SerGly Thr Ser Met Ala Thr Pro His Val Ala Gly Val Ala 210 215 220 Ala LeuVal Lys Ser Arg Tyr Pro Ser Tyr Thr Asn Asn Gln Ile Arg 225 230 235 240Gln Arg Ile Asn Gln Thr Ala Thr Tyr Leu Gly Ser Pro Ser Leu Tyr 245 250255 Gly Asn Gly Leu Val His Ala Gly Arg Ala Thr Gln 260 265 3 275 PRTBacillus 3 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro AlaLeu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala ValIle Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala GlyGly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn AsnSer His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn SerIle Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala ValLys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile AsnGly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn MetSer Leu Gly Gly 115 120 125 Pro Ser Pro Ser Ala Ala Leu Lys Ala Ala ValAsp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala GlyAsn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr ProGly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser SerAsn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp ValMet Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn LysTyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp ThrAsn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 LeuGly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270Ala Ala Gln 275 4 274 PRT Bacillus 4 Ala Gln Thr Val Pro Tyr Gly Ile ProLeu Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly AlaAsn Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His ProAsp Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala TyrAsn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val AlaAla Leu Asp Asn Thr Thr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser ValSer Leu Tyr Ala Val Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Thr TyrSer Gly Ile Val Ser Gly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly MetAsp Val Ile Asn Met Ser Leu Gly Gly Pro 115 120 125 Ser Gly Ser Thr AlaMet Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val ValVal Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn 145 150 155 160 Thr AsnThr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 GlyAla Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200205 Pro Thr Ser Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala ThrTyr Leu 245 250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn ValGlu Ala Ala 260 265 270 Ala Gln 5 275 PRT Bacillus 5 Ala Gln Ser Val ProTyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln GlyTyr Thr Gly Ser Asn Val Lys Val Ala Val Leu Asp 20 25 30 Ser Gly Ile AspSer Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val AlaSer Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His ValAla Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu GlyVal Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser ThrGly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp AlaIle Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 ProThr Gly Ser Ala Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140Ser Gly Ile Leu Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150155 160 Ser Ser Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser SerAla 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile GlnSer Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr SerMet Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu SerLys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg LeuGlu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly LysGly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 6 1143 DNABacillus 6 atgaagaaac cgttggggaa aattgtcgca agcaccgcac tactcatttctgttgctttt 60 agttcatcga tcgcatcggc tgctgaagaa gcaaaagaaa aatatttaattggctttaat 120 gagcaggaag ctgtcagtga gtttgtagaa caagtagagg caaatgacgaggtcgccatt 180 ctctctgagg aagaggaagt cgaaattgaa ttgcttcatg aatttgaaacgattcctgtt 240 ttatccgttg agttaagccc agaagatgtg gacgcgcttg aactcgatccagcgatttct 300 tatattgaag aggatgcaga agtaacgaca atggcgcaat cagtgccatggggaattagc 360 cgtgtgcaag ccccagctgc ccataaccgt ggattgacag gttctggtgtaaaagttgct 420 gtcctcgata caggtatttc cactcatcca gacttaaata ttcgtggtggcgctagcttt 480 gtaccagggg aaccatccac tcaagatggg aatgggcatg gcacgcatgtggccgggacg 540 attgctgctt taaacaattc gattggcgtt cttggcgtag cgccgagcgcggaactatac 600 gctgttaaag tattaggggc gagcggttca ggttcggtca gctcgattgcccaaggattg 660 gaatgggcag ggaacaatgg catgcacgtt gctaatttga gtttaggaagcccttcgcca 720 agtgccacac ttgagcaagc tgttaatagc gcgacttcta gaggcgttcttgttgtagcg 780 gcatctggga attcaggtgc aggctcaatc agctatccgg cccgttatgcgaacgcaatg 840 gcagtcggag ctactgacca aaacaacaac cgcgccagct tttcacagtatggcgcaggg 900 cttgacattg tcgcaccagg tgtaaacgtg cagagcacat acccaggttcaacgtatgcc 960 agcttaaacg gtacatcgat ggctactcct catgttgcag gtgcagcagcccttgttaaa 1020 caaaagaacc catcttggtc caatgtacaa atccgcaatc atctaaagaatacggcaacg 1080 agcttaggaa gcacgaactt gtatggaagc ggacttgtca atgcagaagcggcaacacgc 1140 taa 1143 7 1086 DNA Bacillus 7 atgagacaaa gtctaaaagttatggttttg tcaacagtgg cattgctttt catggcaaac 60 ccagcagcag caggcggggagaaaaaggaa tatttgattg tcgtcgaacc tgaagaagtt 120 tctgctcaga gtgtcgaagaaagttatgat gtggacgtca tccatgaatt tgaagagatt 180 ccagtcattc atgcagaactaactaaaaaa gaattgaaaa aattaaagaa agatccgaac 240 gtaaaagcca tcgaagagaatgcagaagta accatcagtc aaacggttcc ttggggaatt 300 tcattcatta atacgcagcaagcgcacaac cgcggtattt ttggtaacgg tgctcgagtc 360 gctgtccttg atacaggaattgcttcacac ccagacttac gaattgcagg gggagcgagc 420 tttatttcaa gcgagccttcctatcatgac aataacggac acggaactca cgtggctggt 480 acaatcgctg cgttaaacaattcaatcggt gtgcttggtg tacgaccatc ggctgacttg 540 tacgctctca aagttcttgatcggaatgga agtggttcgc ttgcttctgt agctcaagga 600 atcgaatggg caattaacaacaacatgcac attattaata tgagccttgg aagcacgagt 660 ggttctagca cgttagagttagctgtcaac cgagcaaaca atgctggtat tctcttagta 720 ggggcagcag gtaatacgggtagacaagga gttaactatc ctgctagata ctctggtgtt 780 atggcggttg cagcagttgatcaaaatggt caacgcgcaa gcttctctac gtatggccca 840 gaaattgaaa tttctgcacctggtgtcaac gtaaacagca cgtacacagg caatcgttac 900 gtatcgcttt ctggaacatctatggcaaca ccacacgttg ctggagttgc tgcacttgtg 960 aagagcagat atcctagctatacgaacaac caaattcgcc agcgtattaa tcaaacagca 1020 acgtatctag gttctcctagcctttatggc aatggattag tacatgctgg acgtgcaaca 1080 caataa 1086 8 38 DNABacillus 8 cacagtatgg gcgcagggct tgacattgtc gcaccagg 38 9 28 DNABacillus 9 gtatggcgca gagctcgaca tttgtcgc 28 10 30 DNA Bacillus 10cacagtatgg gcgcagggct tgacattgtc 30 11 23 DNA Bacillus 11 caatgtcaagatctgcgcca tac 23 12 36 DNA Bacillus 12 agcttaaacg gtacatcgat ggctactcctcatgtt 36 13 24 DNA Bacillus 13 acggtacatc gtgcgctact cctc 24 14 36 DNABacillus 14 agcttaaacg gtacatcgat ggctactcct catgtt 36 15 22 DNABacillus 15 cggtacatcg gcggctactc ct 22 16 27 DNA Bacillus 16 cttgtagcggcatctgggaa ttcaggt 27 17 18 DNA Bacillus 17 attcccagct gccgctac 18 18 27DNA Bacillus 18 tatgccagct taaacggtac atcgatg 27 19 23 DNA Bacillus 19cgatgtaccg gataagctgg cat 23 20 24 DNA Bacillus 20 tgtggcccgg gacgattgctgctt 24 21 24 DNA Bacillus 21 aagcagcaat gtcccccggc caca 24 22 32 DNABacillus 22 cagcttaaac ggtacatcga tggctactcc tc 32 23 32 DNA Bacillus 23gaggagtagc acacgatgta cagtttaagc tg 32

What is claimed is:
 1. An isolated polynucleotide encoding subtilisin309 having an amino acid sequence which comprises the sequence of SEQ IDNO:
 1. 2. The polynucleotide of claim 1 having a nucleic acid sequencewhich comprises the sequence of nucleotides 334-1140 of SEQ ID NO:
 6. 3.The polynucleotide of claim 2 having a nucleic acid sequence whichcomprises the sequence of nucleotides 82-1140 of SEQ ID NO:
 6. 4. Thepolynucleotide of claim 3 having a nucleic acid sequence which comprisesthe sequence of nucleotides 1-1140 of SEQ ID NO:
 6. 5. A nucleic acidconstruct comprising the polynucleotide of claim 1 operably linked toone of more control sequences that direct the production of subtilisin309 in a suitable expression host.
 6. A recombinant expression vectorcomprising the nucleic acid construct of claim 5, a promoter, andtranscriptional and translational stop signals.
 7. A host cell in whichthe nucleic acid construct of claim 5 has been introduced.
 8. The hostcell of claim 7, which is a Bacillus cell.
 9. The host cell of claim 8,which is a Bacillus subtilis cell.
 10. A method for producing subtilisin309 comprising (a) cultivating the host cell of claim 7 under conditionssuitable for production of subtilisin 309; and (b) recovering subtilisin309.
 11. The method of claim 10, wherein the host cell is a Bacilluscell.
 12. The method of claim 11, wherein the host cell is a Bacillussubtilis cell.
 13. The method of claim 11, wherein the nucleic acidconstruct comprises a nucleic acid sequence comprising the sequence ofnucleotides 334-1140 of SEQ ID NO:
 6. 14. The method of claim 13,wherein the nucleic acid construct comprises a nucleic acid sequencecomprising the sequence of nucleotides 82-1140 of SEQ ID NO:
 6. 15. Themethod of claim 14, wherein the nucleic acid construct comprises anucleic acid sequence comprising the sequence of nucleotides 1-1140 ofSEQ ID NO: 6.